Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety

Abstract

Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animal-derived feeder layers and media containing animal-derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-the-shelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process.

A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use

“…Representative overlay histograms showed no difference in the positive staining for (a) the proliferation marker cytokeratin 14 (K14) and (c) the wound healing marker cytokeratin 6 (K6) between AK in different culture conditions. Anova all pair-wise comparison showed no statistically significant differences in the percentage of positive events of (b) K14 and (d) K6 between the different culture conditions (n = 3, p = 0.05) towards developing and testing defined medium conditions (Boyce and Ham 1983;Sun et al 2004;Coolen et al 2007;Mujaj et al 2010;De Corte et al 2011). In these experiments we did not test defined medium conditions beyond the use of EpiLife with EDGS.…”

Optimization of human keratinocyte culture to develop an artificial human skin model: cell alternatives as feeder layer of Advanced Therapies

“…La optimización en el proceso de expansión celular de los queratinocitos, ha llevado a muchos grupos de investigación a estudiar un feeder alternativo (19) (20), formado por células autólogas con el fin de evitar el rechazo por parte del paciente. En nuestro modelo de piel artificial humana, el tiempo limitado en cultivo, hace imposible el uso de un feeder autólogo.…”

Keratinocyte Culture Techniques in Medical and Scientific Applications

“…In order to replace autografts a number of approaches have been developed, essentially they are either based on the use of acellular matrices, transfer of cultured cells or a combination of both. Since the 80ties of the last century the use of autologous keratinocytes has been reported by different burn centres and established as an alternative for severely burned patients with little donor skin (De Corte et al, 2011). Keratinocyte transplantation after burns and in chronic wound treatment is potentially useful in clinical practice to improve functional outcome in burn patients.…”

“…Another potential problem for clinical application of cultured human keratinocytes is the use of serum because of safety issues. Other companies offer recombinant growth factors and enzymes for animal-free production of cell therapeutics like trypsin-like dislodging enzyme (TrypLE, Gibco, Invitrogen) (De Corte et al, 2011).…”